This renewal application proposes to continue our work to develop the multiple displacement amplification (MDA) reaction which is used for whole genome amplification. MDA can be used to obtain amplified DNA from single cells for use in sequencing. The study will continue to focus on microbes inhabiting the human body. New methods will be demonstrated for amplifying DNA from single cells of uncultured bacteria obtained from human clinical specimens including skin, throat and fecal samples. Basic research will continue into the enzymology of the MDA reaction in order to reduce amplification bias. Microcolony technology will also be used to reduce amplification bias by providing DNA template from multiple cells rather than a single cell. Research will continue into better methods to isolate individual cell by flow cytometry and micromanipulation. Fluorescent in situ hybridization (FISH) probes will be tested for isolating specific microbes of interest. These methods will be introduced into our program to supply amplified genomic DNA for sequencing by the NIH Human Microbiome Project. There will also be increased emphasis on medically important questions including analysis of the normal bacterial community colonizing humans and pathogens involved in hospital acquired infections. We will demonstrate single cell methods for investigating a GI tract pathogen, C. difficile, that causes hospital acquired infections (in collaboration with the University of California at San Diego Medical Center). Methods to sequence uncultivated viruses and eukaryotes will also be introduced. Improved informatic methods to assemble sequences derived from MDA reactions will be developed in collaboration with Illumina Inc. and Pavel Pevzner, UCSD.
This renewal application continues our work to develop the multiple displacement amplification (MDA) reaction which is used to amplify DNA. MDA can be used to obtain amplified DNA from single cells for use in sequencing. More than one billion copies of DNA are produced starting with the single DNA copy contained in a bacterial cell. This study will continue to focus on microbes inhabiting the human body. New methods will be demonstrated for amplifying DNA from single cells of uncultured bacteria obtained from human clinical specimens including skin, throat and fecal samples. For the many bacteria that cannot be grown in culture, MDA will make it possible to obtain enough DNA for use in sequencing their genomes.
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